Morphine and other opioids remain the most frequently prescribed drugs for the treatment of moderate to severe pain, including pain due to cancer or surgery. The overall goal of this study is to elucidate the mechanisms of morphine and other opioid-induced tolerance in the ileum and brain but not in the colon which leads to constipation and limits the chronic use of these excellent pain relievers in man. This proposal represents a collaboration of two senior laboratories, one with expertise in gastrointestinal physiology and the other with extensive experience in studying the acute and chronic effects of morphine and other opioids, utilizing molecular, cellular and whole animal approaches. The main hypothesis to be tested is that differences in the cellular signaling properties between the colon and ileum result in morphine-induced tolerance in the ileum but not the colon. We further propose that the ratio of tolerance to various opioids is the same across these tissues. Our preliminary data demonstrate that as marked tolerance develops to the antinociceptive effects of morphine following chronic administration in mice, tolerance does not develop to its constipating effects. The major objective of the work proposed in year 1 is to elucidate whether this difference in tolerance in different tissues observed for morphine is the same for other opioids both in vivo and in vitro. In the second year we propose to further evaluate our interesting finding that nicotinamide, an endogenous CD-38 inhibitor, induces tolerance to morphine in the colon, a phenomenon not previously reported. We will test the hypothesis that the CD-38 pathway prevents tolerance development in the colon and that its blockade by either chemical means or genetically in knock out animals allows tolerance to occur. We propose to investigate whether CD38 in other tissues such as the ileum or brain causes a regulatory effect on the development of tolerance in these tissues as well. Further, we will investigate whether additional steps in the signal transduction process such as the phosphorylation of mu opioid receptors, the activity of protein kinase C and A, GRK alter opioid tolerance development similarly or differently in various tissues. Our preliminary data demonstrate that inhibition of the CD38 pathway by either nicotinamide or 8-bromo cyclic ADPribose results in the induction of tolerance to morphine in the colon. We will test the effects of nictoinamide, ryanodine and cADPribose on tolerance development and on dependence to morphine in the isolated colonic muscle strips and determine if inhibition of the CD38 pathways results in changes in mu receptor phosphorylation in enteric neurons. The long-term major public health implication of this research is the use of this information to develop medications to treat chronic pain that are devoid of constipation. These studies will also increase our understanding of the potential mechanisms in the development of tolerance and ultimately physical dependence to opioids in the brain.